Protein A is a membrane protein encoded by a bacterium (Staphylococcus aureus). Protein A consists of five homologues domains arranged as E-D-A-B-C. Protein A is unique in its ability to bind to most antibodies (including IgG, IgM, IgE, IgA) from various species including humans. Thus, Protein A has been widely used as a protein ligand immobilized on a solid support (eg., resin) to form an affinity column for antibody purification for research and therapeutic applications.
To fully exploit the antibody-binding property of Protein A for both pharmaceutical and biotechnological applications, methods to improve the immobilization efficiency of Protein A on a solid resin, as well as methods of increasing protein A stability and reducing its leakage after immobilization have been under investigation. In this regard, U.S. Pat. Nos. 5,084,559 and 5,260,373 describe methods of immobilizing Protein A in which a single cysteine residue, or a single arginine residue, is added to the N-terminus of Protein A, or a domain, or multiple domains thereof, to facilitate their immobilization. Methods to stabilize the immobilized protein have also been described in U.S. Pat. No. 7,485,704 in which Protein A ligand leakage is alleviated by reducing temperature during the antibody purification, and in US patent (U.S. Pat. No. 7,709,209) in which alkali stability of a protein A domain is increased by mutating specific asparagine residues in its sequence to an amino acid other than glutamine or aspartic acid.
Despite the foregoing, there remains a need to develop improved methods for protein immobilization and enhanced ligand stability.
Affinity chromatography is one of the most useful separation methods to purify proteins with high recovery rate in a single step. Epoxy-based coupling is a widely used technology for peptide or protein immobilization. The epoxy group contains an oxygen atom joined by single bonds to two adjacent carbon atoms to form an epoxide ring. This functional group is able to react with different nucleophilic groups on a protein surface to form a strong linkage with minimal chemical modifications. However, the immobilization efficiency on epoxy resin is largely dependent on the composition of a given peptide or protein, including amino acid sequence, pI and protein secondary structures, as well as reaction conditions including pH, coupling buffer composition and salt concentration.